This research is intended to further define the physiological roles of nitric oxide (NO) synthesis from L-arginine during hypoxia. The high output immune/inflammatory NO synthase (iNOS) is induced by cytokines when there are perturbations in tissues (microbial invasion, inflammation, and/or mechanical damage) that represent a serious threat to stability and can result in hypoxia. Nitric oxide synthesized by iNOS activates soluble guanylyl cyclase in vascular smooth muscle, causing vessel dilation and increased blood flow, but other roles of iNOS derived NO in the local response to hypoxia have not been considered. The hypothesis is that NO synthesized by iNOS regulates mitochondrial function and these NO mediated changes reorganize mitochondrial and cytosolic energy generating pathways for more efficient function under hypoxic conditions. The hypothesis is that mitochondrial aconitase (m-aconitase) functions in normal cells as a metabolic switch (activatable by NO) which converts the Krebs cycle to a Krebs pathway and changes the mitochondrial energy source from glucose to L-glutamine. This suggests that NO induced reorganization of mitochondrial metabolism in normal cells is similar to the pattern of metabolic organization which is termed the malignant biochemical phenotype. This metabolic mode could equip normal and malignant cells for efficient function under hypoxic conditions. The metabolic consequences of NO induced inhibition of m-aconitase in normal cells will be compared, using a polarographic assay system, to results obtained in parallel experiments carried out on a cell line that has a fully developed malignant biochemical phenotype. The hypothesis is that inhibition of a second mitochondrial enzyme (cytochrome oxidase) by NO regulates energy metabolism and O2 consumption under hypoxic conditions in both normal and malignant cells. Competitive inhibition of cytochrome oxidase by NO preserves a limited supply of O2 under hypoxic conditions. This prevents the generation of an anaerobic microenvironment by unregulated mitochondrial O2 consumption during hypoxia. The diminished supply of O2 remains available for other enzymatic reactions (e.g, iNOS, other NOS isoforms, and other O2 dependent reactions) needed to reverse hypoxia. The hypothesis will be tested, using a gas chromatography-mass spectrometry assay, that cells in culture or isolated mitochondria, like purified cytochrome oxidase, reduce NO to N2O under hypoxic or anoxic conditions. The consumption of NO by cytochrome oxidase could rapidly reverse NO mediated inhibition. The hypothesis also suggests that NO, by acting as an alternative electron receptor, supports limited ("pilot light" level) oxidative phosphorylation facilitated by the reduction of No to N2O.